(i) Field of the Invention
The present invention relates to potato starches having an amylose content of less than 10% by weight, a phosphate content in the C6 position of between 35 and 100 nmol of phosphate per milligram of starch and an increased content of side chains having a DP of from 12 to 19 as compared with the potato starch from corresponding wild-type potato plants.
(ii) Description of the Related Art
In view of the increasing importance which is currently being attached to plant components as renewable sources of raw material, one of the tasks of biotechnological research is to endeavor to adapt these plant raw materials to the requirements of the processing industry. In addition to this, it is necessary to achieve a great diversity of substances in order to enable renewable raw materials to be used in as many areas of employment as possible.
While the polysaccharide starch is composed of chemically uniform basic units, i.e. the glucose molecules, it is a complex mixture of different molecular forms which exhibit differences with regard to the degree of polymerization and branching and consequently differ greatly from each other in their physicochemical properties. A distinction is made between amylose starch, an essentially unbranched polymer composed of alpha-1,4-glycosidically linked glucose units, and amylopectin starch, a branched polymer in which the branches are formed as a result of the appearance of additional alpha-1,6-glycosidic linkages. Another important difference between amylose and amylopectin lies in their molecular weights. While amylose, depending on the origin of the starch, has a molecular weight of 5×105-106 Da, the molecular weight of amylopectin is between 107 and 108 Da. The two macromolecules can be differentiated by their molecular weight and their different physicochemical properties, something which can most readily be visualized by their different iodine-binding properties.
Amylose was regarded for a long time as being a linear polymer which consisted of alpha-1,4-glycosidically linked alpha-D-glucose monomers. However, more recent studies have demonstrated the presence of a small proportion of alpha-1,6-glycosidic branching points (approx. 0.1%) (Hizukuri and Takagi, Carbohydr. Res. 134 (1984), 1-10; Takeda et al., Carbohydr. Res. 132, (1984), 83-92).
Amylopectin constitutes a complex mixture composed of glucose chains which are branched differently. Amylopectin is more strongly branched than amylose. Side chains are linked by way of α-1,6-glycosidic bonds to the main chain, which is composed of α-1,4-glycosidically linked α-D-glucose monomers. According to textbook data (Voet and Voet, Biochemistry, John Wiley & Sons, 1990), the α-1,6 branches occur every 24 to 30 glucose residues on average. This corresponds to a degree of branching of approx. 3%-4%. The data with regard to the degree of branching are variable and depend on the origin (e.g. plant species, plant variety, etc.) of the given starch. In typical plants which are used for industrial starch production, e.g. corn, wheat or potato, approx. 20%-30% of the synthesized starch is composed of amylose starch and approx. 70%-80% is composed of amylopectin starch.
The functional properties, such as the solubility, the retrogradation behavior, the ability to bind water, the film-forming properties, the viscosity, the pasting properties, the freeze/thaw stability, the acid stability, the gel strength and the starch grain size of starches are influenced, inter alia, by the amylose/amylopectin ratio, the molecular weight, the pattern of side chain distribution of the amylopectin, the content of ions, the content of lipid and protein, the mean starch grain size, the starch grain morphology, etc. The functional properties of starch are also influenced by the content of phosphate, in the starch. In this connection, a distinction is made between phosphate which is covalently bonded in the form of monoesters to the glucose molecules of the starch (termed starch phosphate below) and phosphate in the form of phospholipids which are associated with the starch.
The content of starch phosphate varies in dependence on the plant type. Thus, for example, certain corn mutants synthesize a starch having an elevated content of starch phosphate (waxy corn 0.002% and high-amylose corn 0.013%) whereas conventional corn types only exhibit traces of starch phosphate. Small quantities of starch phosphate are also found in wheat (0.001%) whereas it has not been possible to detect any starch phosphate in oats and sorghum. Relatively large quantities of starch phosphate have thus far been detected in tuber or root storage starch, for example tapioca (0.008%), sweet potato (0.011%), arrowroot (0.021%) or potato (0.089%).
The percentage values of the starch phosphate content which have been cited above in each case relate to the dry weight of the starch and were determined by Jane et al. (1996, Cereal Foods World 41 (11), 827-832). Starch phosphate can be present in the form of monoesters at the C2, C3 or C6 position in the polymerized glucose monomers (Takeda and Hizukuri, 1971, Starch/Stärke 23, 267-272). In general, from about 30% to 40% of the covalently bound starch phosphate groups are located in the C3 position, and from about 60% to 70% are located in the C6 position, in the glucose monomers (Blennow et al., 2000, Int. J. of Biological Macromolecules 27, 211-218).
Potato amylopectin starches, i.e. starches having an amylopectin content of more than 90% and an amylose content of less than 10%, can be obtained from potato plants in which the activity of the starch granule-bound starch synthase GBSSI (“Granule-Bound Starch Synthase I”) is reduced (Shure et al., 1983, Cell 35, 225-233; Hovenkamp-Hermelink et al., 1987, Theoretical and Applied Genetics 75, 217-221; Visser et al., 1991, Mol. Gen. Genet. 225, 289-296). GBSSI is involved in the formation of amylose. Inhibition of the GBSSI activity leads to the synthesis of starch which is almost exclusively composed of amylopectin. The corresponding GBSSI gene in maize is known under the name “waxy”. Amylopectin starches are also termed waxy starches.
Plants in which the activity of soluble starch synthase III (SSIII) is reduced have also been described (Abel et al., 1996, The Plant Journal 10(6), 981-991; Lloyd et al., 1999, Biochemical Journal 338, 515-521). As compared with starch which is isolated from corresponding wild-type plants, starch from these plants exhibits a relative shift of the amylopectin side chains from relatively long chains to short chains (Lloyd et al., 1999, Biochemical Journal 338, 515-521), an increased content of phosphate, no change in the amylose content (Abel et al., 1996, The Plant Journal 10(6), 9891-9991) and a reduced final viscosity in the RVA analysis (Abel, 1995, Berlin Free University dissertation).
Plants in which the activity of branching enzyme I (BEI) is reduced have also been described (Kossmann et al., 1991, Mol. Gen. Genet. 230, 39-44; Safford et al., 1998, Carbohydrate Polymers 35, 155-168; WO 92/14827). Safford et al. (1998, see above) report that corresponding potatoes produce a starch which has a slightly altered amylose/amylopectin ratio. Nor does the degree of branching of the amylopectin differ significantly from that of a starch which is isolated from wild-type potatoes. However, the starch-bound phosphate content is slightly increased.
WO 01/19975 describes plants in which the GBSSI and the SSII and/or SSIII activities are reduced. As compared with starch from wild-type potatoes, starch from potatoes having reduced activities of GBSSI, SSII and SSIII exhibit a lower amylose content, altered swellability and pasting properties and higher freeze/thaw stability.
WO 01/12782 describes plants in which both the GBSSI activity and the BEI activity are reduced. Starch from these potato plants exhibits a reduced amylose content as compared with potato starch from wild-type plants and an elevated phosphate content and/or a reduced pasting temperature in the RVA analysis as compared with potato starch from plants having the waxy phenotype.
WO 00/08184 describes, inter alia, plants in which both the SSIII activity and the BEI activity are reduced. Starch from these plants exhibits a markedly elevated phosphate content as compared with starch from wild-type plants.